The overall program project will test the hypothesis that a combination of thymidine and select antifolates directed at the thymidylate synthase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) can be potent and safe antimalarial agents, worthy of future clinical trials in humans. Blood-stage forms of malaria parasites are completely dependent on de novo pyrimidine biosynthesis and fail to salvage preformed pyrimidines, unlike human cells. Of all the enzymes available in de novo pyrimidine biosynthesis, DHFR-TS is of particular interest. Inhibitors of the DHFR domain are proven drug targets in malaria. The PI's laboratory has previously demonstrated that one TS antifolate (1843U89) inhibits Plasmodium TS with a Ki of 1 nM and inhibits parasite proliferation with an ECs0 of 70 nM. With 10 uM thymidine, mammalian cells show no toxicity to this compound, even at 10,000 times higher concentrations. Starting with this very strong lead, we will develop additional compounds that are even more potent. There are opportunities to derive potent TS antifolates from more than one chemical scaffold. To avert drug resistance, we are particularly interested in compounds which do not require polyglutamylation for optimum TS binding or folate Iransporters to enter cells. The crystal structure of malaria TS is now available and global oncology programs have generated a large aumber of TS inhibitors from multiple chemical classes. Compounds that are in clinical trials, and close 5erivatives, are immediately applicable for "piggy-back" strategies against malaria. Based on preliminary data i'om lead compounds, modeling and NMR-based ligand discovery initiatives, about 50-00 potential TS antifolates per year will be synthesized. This Project will measure the interactions of these new TS antifolates with the target enzyme TS and enzymes which metabolize antifolates. In addition, using P. falciparum in culture, the antiproliferative activity of the TS inhibitors will be evaluated as well as the ease with which parasites acquire resistance to these molecules. Finally, in a validation study, biochemical and genetic approaches will test the mode of action of the candidate TS inhibitors. The best antimalarials will be passed on for safety and efficacy testing.